Extracellular matrix material conduits and methods of making and using same

ABSTRACT

Extracellular matrix (ECM) material conduits are disclosed. Methods for regenerating atrioventricular valves to replace defective atrioventricular valves within a heart of a subject using the ECM material conduits are also disclosed. Methods of sterilizing and decellularizing an ECM material are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S.Provisional Patent Application No. 61/490,693, filed on May 27, 2011,U.S. Provisional Patent Application No. 61/490,873, filed on May 27,2011, U.S. Provisional Patent Application No. 61/491,723, filed on May31, 2011, and U.S. Provisional Patent Application No. 61/650,911, filedon May 23, 2012, each of which is hereby incorporated by referenceherein in its entirety.

FIELD

The invention relates to extracellular matrix (ECM) material conduitsand methods of using such ECM material conduits to regenerateatrioventricular (AV) valves within a heart of a subject.

BACKGROUND

There are many known types of replacement heart valves. The selection ofa particular type of replacement heart valve depends on factors such asthe location of the valve, the age and other specifics of the patient,and the surgeon's experiences and preferences. Commonly used replacementheart valves can be classified in the following three groups: mechanicalvalves; allograft tissue valves; and xenograft tissue valves.

Mechanical heart valves, including, for example and without limitation,caged-ball valves, bi-leaflet valves, and tilting disk valves aretypically attached to a sewing ring so that the valve prosthesis can besutured to the patient's native tissue to hold the mechanical valve inplace postoperatively. Although mechanical heart valves haveadvantageous long-term durability, these mechanical valves also have apropensity to cause the formation of blood clots in a patient. If suchblood clots form on the mechanical valve, they may preclude the valvefrom opening or closing correctly or, more importantly, may disengagefrom the valve and embolize to the brain, causing an embolic stroke.Thus, the patients who receive such mechanical valves are typicallyrequired to take systemic anticoagulant drugs for the rest of theirlives. In addition to being expensive, these anticoagulant drugs canthemselves be dangerous in that they can cause abnormal bleeding in thepatient that can lead to a hemorrhagic stroke.

Allograft tissue valves are harvested from human sources, such as humancadavers. Unlike mechanical heart valves, allograft tissue valvestypically do not promote blood clot formation and, therefore, avoid theneed for prescribing an anticoagulant medication for the patient.However, allograft tissue valves are not available in sufficient numbersto satisfy the needs of all patients who need new heart valves.Furthermore, there have been significant complications when allografttissue valves have been used to replace atrioventricular (AV) valveswithin a subject. Moreover, allograft tissue valves can be moredifficult to implant than mechanical valves or xenograft valves. Becauseof these difficulties in implantation, the operative risk associatedwith allograft tissue valves is often greater than the operative risksassociated with mechanical valves and xenograft valves.

Xenograft tissue valves are formed from non-human tissue sources, suchas cows or pigs. Most known xenograft tissue valves are constructed bysewing and/or constructing valve leaflets from a non-human tissue sourceand then securing the leaflets within a patient's heart using a stentand/or a sewing ring. These xenograft tissue valves are less likely tocause blood clot formation than comparable mechanical valves, andtherefore, patients that receive xenograft tissue valves are not alwaysrequired to take anticoagulant medications. However, xenograft tissuevalves are prone to calcification and lack the long-term durability ofmechanical valves and, consequently, require frequent replacement ascompared to mechanical valves. One factor that may contribute to thesefailures is the chemical treatment that the xenograft tissue valvestypically undergo to reduce antigenicity of the animal tissue. Withoutthese chemical treatments, xenograft tissue valves can trigger an immuneresponse in a patient, which can lead to rejection of the tissue valveby the patient. Another factor that may contribute to the lack ofdurability of the xenograft tissue valves is the presence of a stentand/or sewing ring, which can prevent the xenograft tissue valve fromaccurately approximating the anatomy of a normal heart valve.

Known tissue conduits, including those described in U.S. Pat. Nos.5,480,424 and 5,713,950, both of which are expressly incorporated hereinby reference in their entirety, suffer from various limitations,including many of the limitations of known xenograft tissue valves. Forexample, known tissue conduits suffer from antigenicity of the conduits,which is typically addressed using chemical treatments that lessenpost-implantation durability of the conduit. Additionally, these knownconduits are rapidly degraded within a patient's heart such that theycan only serve as competent heart valve replacements for a matter ofmonths.

Thus, what is needed in the art is a readily available, highly durable,and affordable tissue prosthesis that can be easily implanted toregenerate an anatomically accurate AV valve within the heart of asubject. There is a further need in the art for a sterile, acellulartissue prosthesis that can be implanted to regenerate an AV valve withinthe heart of a subject.

SUMMARY

Extracellular matrix (ECM) material conduits are disclosed. In oneaspect, a disclosed ECM material conduit defines a lumen and has aninlet portion and an outlet portion. The inlet portion of the ECMmaterial conduit includes an inlet of the lumen. The outlet portion ofthe ECM material conduit includes an outlet of the lumen. The inletportion and the outlet portion of the ECM material conduit can each havean outer circumference. The ECM material conduit is sterile andacellular. Methods of regenerating an atrioventricular (AV) valve toreplace a defective AV valve within a heart of a subject are alsodisclosed. In one aspect, the methods include removing the defective AVvalve from the heart of the subject. The methods also include implantingan ECM material conduit within the heart of the subject to regenerate afunctional AV valve.

BRIEF DESCRIPTION OF THE FIGURES

These and other features of the preferred embodiments of the inventionwill become more apparent in the detailed description in which referenceis made to the appended drawings wherein:

FIG. 1 is a cut-away view of a human heart.

FIG. 2 is a perspective view of an ECM material conduit as it isattached to an annulus of an atrioventricular valve and to two papillarymuscles, as described herein.

FIG. 3 is a bottom perspective view of the ECM material conduit shown inFIG. 2. The ECM material conduit depicted in FIG. 3 is attached to afirst papillary muscle at a first attachment point and to a secondpapillary muscle at a second attachment point.

FIG. 4 is an image of a native tri-cuspid valve following removal fromthe heart of a subject. FIG. 4 includes markings corresponding toparticular measurements that were performed on various native porcineand human tri-cuspid valves.

FIG. 5 is an image of an ECM material conduit following implantation ofthe ECM material conduit following removal of a native tri-cuspid valve.FIG. 5 displays the ECM material conduit in a closed position within anin vitro model of the right heart.

FIG. 6 depicts Doppler echocardiography images taken postoperatively foran exemplary ECM material conduit functioning as a tri-cuspid valvewithin an animal. FIG. 6( a) depicts the ECM material conduit one monthfollowing the operation with the valve in a closed position. FIG. 6( b)depicts the ECM material conduit immediately post-operatively with thevalve in an open position. FIG. 6( c) depicts the ECM material conduitimmediately post-operatively with the valve in a closed position.

FIG. 7 displays images of a regenerated tri-cuspid valve at various timepoints following implantation of an exemplary ECM material conduit asdescribed herein. FIG. 7( a) shows regeneration at 3 months. FIG. 7( b)shows regeneration at 5 months. FIG. 7( c) shows regeneration at 8months. FIG. 7( d) shows regeneration at 12 months.

FIG. 8 displays an image of a regenerated tri-cuspid valve at threemonths following implantation of an exemplary ECM material conduit asdescribed herein.

FIGS. 9-10 depict the results of an experiment in which DNA content wasmeasured for small intestinal submucosa (SIS) compositions followingvarious sterilization methods, including the sterilization methodsdescribed herein. FIG. 9 shows the DNA content of each SIS compositionfollowing sterilization. FIG. 10 shows the percentage of DNA that wasremoved from each SIS composition following sterilization, as comparedto raw, unprocessed SIS.

FIGS. 11-12 depict the results of an experiment in which native growthfactor content was measured for SIS compositions following varioussterilization methods, including the sterilization methods describedherein. FIG. 11 shows the bFGF content of each SIS composition(normalized by dry weight of samples) following sterilization. FIG. 12shows the active TGF-β content of each SIS composition (normalized bydry weight of samples) following sterilization.

FIG. 13 depicts the results of an experiment in which bFGF wasincorporated into SIS compositions during rapid depressurization, asdescribed herein. FIG. 13 shows the bFGF content for each SIScomposition (normalized by dry weight of samples) following rapiddepressurization.

FIG. 14 depicts the results of an experiment in which the tensilestrength of two-ply SIS compositions was measured following varioussterilization methods, including the sterilization methods describedherein. FIG. 14 shows the tensile strength measured for each SIScomposition following sterilization.

FIG. 15 depicts the results of an experiment in which native growthfactor content was measured for SIS compositions following varioussterilization and/or decellularization methods, including thesterilization and decellularization methods described herein. FIG. 15shows the bFGF enzyme-linked immunosorbent assay (ELISA) results foreach SIS composition (normalized by dry weight of samples) followingsterilization and/or decellularization.

FIG. 16 shows the DNA content in SIS after it is processed in variousways. The baseline measurement is raw. The tissue was then exposed tosupercritical CO₂ followed by rapid depressurization (RDP) to facilitateenhanced removal of DNA and cellular debris. After the RDP, the tissuewas placed in supercritical CO₂ with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 17 shows the Percent removal of DNA from SIS after it is processedin various ways. The baseline measurement is raw. The tissue was thenexposed to supercritical CO₂ followed by rapid depressurization (RDP) tofacilitate enhanced removal of DNA and cellular debris. After the RDP,the tissue was placed in supercritical CO₂ with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 18 shows the variable active Transforming Growth Factor (TGF-beta)content in SIS after it is processed in various ways. The baselinemeasurement is raw, or unprocessed SIS followed by processing with onlyTriton X-100 (TX-100) detergent. The tissue was then exposed tosupercritical CO₂ followed by rapid depressurization (RDP) to facilitateenhanced removal of DNA and cellular debris. After the RDP, the tissuewas placed in supercritical CO₂ with peracetic acid (PAA) forsterilization. The comparison is to processed SIS either unsterilized orsterilized with ethylene oxide (ETO).

FIG. 19 shows the variable basic Fibroblast Growth Factor (bFGF) contentin SIS after it is processed in various ways. The baseline measurementis raw, or unprocessed SIS followed by processing with only Triton X-100(TX-100) detergent. The tissue was then exposed to supercritical CO₂followed by rapid depressurization (RDP) to facilitate enhanced removalof DNA and cellular debris. After the RDP, the tissue was placed insupercritical CO₂ with peracetic acid (PAA) for sterilization. Thecomparison is to processed SIS either unsterilized or sterilized withethylene oxide (ETO).

FIG. 20 shows the addition of basic Fibroblast Growth Factor (bFGF)content to SIS using rapid depressurization. The baseline measurement israw, or unprocessed SIS. The comparison is to processed SIS eitherunsterilized or sterilized with ethylene oxide (ETO).

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description, examples, and claims, and their previousand following description. However, before the present devices, systems,and/or methods are disclosed and described, it is to be understood thatthis invention is not limited to the specific devices, systems, and/ormethods disclosed unless otherwise specified, as such can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “attachmentpoint” can include two or more such attachment points unless the contextindicates otherwise.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

As used herein, the terms “optional” and “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat an order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

Without the use of such exclusive terminology, the term “comprising” inthe claims shall allow for the inclusion of any additionalelement—irrespective of whether a given number of elements is enumeratedin the claim, or the addition of a feature could be regarded astransforming the nature of an element set forth in the claims. Except asspecifically defined herein, all technical and scientific terms usedherein are to be given as broad a commonly understood meaning aspossible while maintaining claim validity.

As used herein, a “subject” is an individual and includes, but is notlimited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, abird, a reptile or an amphibian. The term does not denote a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are intended to be included. A “patient” is asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. As used herein, the term“subject can be used interchangeably with the term “patient.”

As used herein, the term “circumference” refers to the perimeter of, orthe length measurement of the boundary defined by, a closed planarfigure. Optionally, as used herein, a “circumference” can correspond tothe perimeter of a closed planar circle. However, it is contemplatedthat a “circumference” can correspond to the perimeter of any closedplanar figure, such as, for example and without limitation, an oval,square, rectangular, trapezoidal, or nonsymmetrical closed planarfigure. For example, as used herein, an outer “circumference” of aconduit corresponds to the perimeter of the closed planar figure definedby an outer surface of the conduit at a particular location along thelongitudinal axis of the conduit.

As used herein, the term “frusto-conical” refers to the shape of aconical frustum, which corresponds to the shape of a cone that has hadits tip truncated by a plane parallel to its base. Thus, as used herein,a “frusto-conical” conduit has a substantially circular cross-sectionthat varies in diameter along its longitudinal axis. The“frusto-conical” conduits disclosed herein have inlet portions andoutlet portions that each have outer circumferences. Optionally, theouter circumference of the outlet portion of a disclosed“frusto-conical” conduit can be greater than the outer circumference ofthe inlet portion of the “frusto-conical” conduit. Alternatively, theouter circumference of the outlet portion of a disclosed“frusto-conical” conduit can be less than the outer circumference of theinlet portion of the “frusto-conical” conduit. In exemplary aspects, theouter circumference of the outlet portion of a disclosed“frusto-conical” conduit can be substantially equal to the outercircumference of the inlet portion of the “frusto-conical” conduit.

As used herein, the term “acellular” is meant to describe extracellularmatrix compositions that are at least 80% decellularized such that theextracellular matrix composition is at least 80% without cells and/orcellular remnants. In some exemplary aspects described herein, the term“acellular” can refer to extracellular matrix compositions that are atleast 90% decellularized such that the extracellular matrix compositionis at least 90% without cells and/or cellular remnants. In otherexemplary aspects described herein, the term “acellular” can refer toextracellular matrix compositions that are at least 95% decellularizedsuch that the extracellular matrix composition is at least 95% withoutcells and/or cellular remnants. In other exemplary aspects describedherein, the term “acellular” can refer to extracellular matrixcompositions that are at least 96% decellularized such that theextracellular matrix composition is at least 96% without cells and/orcellular remnants. In still other exemplary aspects described herein,the term “acellular” can refer to extracellular matrix compositions thatare at least 97% decellularized such that the extracellular matrixcomposition is at least 97% without cells and/or cellular remnants. Infurther exemplary aspects described herein, the term “acellular” canrefer to extracellular matrix compositions that are at least 98%decellularized such that the extracellular matrix composition is atleast 98% without cells and/or cellular remnants. In still furtherexemplary aspects described herein, the term “acellular” can refer toextracellular matrix compositions that are at least 99% decellularizedsuch that the extracellular matrix composition is at least 99% withoutcells and/or cellular remnants. Thus, as used herein, the term“acellular” can refer to extracellular matrix compositions that aredecellularized at levels of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and anypercentages falling between these values.

As used herein, the term “additive” refers to materials that can beselectively incorporated into the disclosed ECM materials to impartpredetermined properties to the sterilized, acellular ECM compositionsdisclosed herein. Such additives can include, for example and withoutlimitation, growth factors, cytokines, proteoglycans, glycosaminoglycans(GAGs), proteins, peptides, nucleic acids, small molecules, cells andpharmaceutical agents, such as statin drugs, corticosterioids,anti-arrhythmic drugs, nonsteroidal anti-inflammatory drugs, otheranti-inflammatory compounds, nanoparticles, and metallic compounds.

As used herein, the term “contemporaneously” refers to the simultaneousand/or overlapping occurrence of events, as well as the sequentialoccurrence of events within thirty minutes before or after one another.Thus, if a first event occurs, then a second event can be said to haveoccurred contemporaneously with the first event if it occurredconcurrently with the first event or within thirty minutes before orafter the first event. For example, if a first method step is performed,then a second method step performed five minutes after the first methodstep can be said to be performed “contemporaneously” with the firstmethod step. Similarly, if the second method step was performed tenminutes before performance of a third method step, then the secondmethod step can be said to be performed “contemporaneously” with thethird method step.

As used herein, the term “supercritical” refers to a fluid state of amaterial when it is held at or above its critical temperature andcritical pressure. When a material is held at or above its criticaltemperature and critical pressure, then it typically adopts functionalproperties of both a gas and a liquid and is said to function as asupercritical fluid. Thus, for example, when carbon dioxide is held ator above its critical temperature (31.1° C.) and its critical pressure(1,071 psi), it behaves as a supercritical carbon dioxide fluid and can,for example, exhibit the expansion properties of a gas while having thedensity of a liquid.

Described herein with reference to FIGS. 1-3 are methods of making andusing extracellular matrix (ECM) material conduits. In one aspect, asdepicted in FIGS. 2-3, an exemplary ECM material conduit 10 can define alumen 12 and have an inlet portion 14 and an outlet portion 18. In thisaspect, it is contemplated that the inlet portion 14 of the ECM materialconduit 10 can comprise an inlet 16 of the lumen 12. It is furthercontemplated that the outlet portion 18 of the ECM material conduit 10can comprise an outlet 20 of the lumen 12.

In additional aspects, the inlet portion 14 and the outlet portion 18 ofan ECM material conduit 10 can each have an outer circumference. In oneaspect, it is contemplated that the outer circumference of the outletportion 18 of the ECM material conduit 10 can be substantially equal tothe outer circumference of the inlet portion 14 of the ECM materialconduit. Optionally, in this aspect, the ECM material conduit 10 can besubstantially cylindrical. In another aspect, it is contemplated thatthe outer circumference of the outlet portion 18 of the ECM materialconduit 10 can be greater than the outer circumference of the inletportion 14 of the ECM material conduit. Optionally, in this aspect, theECM material conduit 10 can be substantially frusto-conical. In afurther aspect, it is contemplated that the outer circumference of theoutlet portion 18 of the ECM material conduit 10 can be less than theouter circumference of the inlet portion 14 of the ECM material conduit.Optionally, in this aspect, the ECM material conduit 10 can besubstantially frusto-conical.

In one aspect, it is contemplated that the ECM material conduit can havea longitudinal axis 24 and a longitudinal length ranging from about 10mm to about 50 mm. In another aspect, it is contemplated that the outercircumferences of the inlet portion 14 and the outlet portion 18 of theECM material conduit 10 can each range from about 25 mm to about 190 mm.Thus, it is further contemplated that, at the inlet portion 14 and theoutlet portion 18 of the ECM material conduit 10, the lumen 12 of theECM material conduit can have a diameter ranging from about 8 mm toabout 60 mm. In an additional aspect, the ECM material conduit 10 canhave a wall 22 having a thickness. In this aspect, it is contemplatedthat the thickness of the wall 22 of the ECM material conduit 10 canrange from about 0.05 mm to about 3.00 mm.

In one exemplary aspect, the outlet portion 18 of the ECM materialconduit 10 can comprise one or more extension portions 15 a, 15 b thatprotrude outwardly from the ECM material conduit 10. It is contemplatedthat the extension portions can be configured to provide an attachmentconfiguration for the papillary muscles that more closely mimics nativefunctionality. It is further contemplated that the extension portions 15a, 15 b can be configured to promote fusion between the native papillarymuscles attached to the ECM material conduit 10 and the regenerated AVvalve formed following implantation of the ECM material conduit asdescribed herein.

In exemplary aspects, when the outlet portion 18 of the ECM materialconduit 10 comprises at least one extension portion as described herein,it is contemplated that one or more of the first, second, and thirdattachment points can be positioned on a corresponding extension portionof the at least one extension portion. In these aspects, it iscontemplated that the at least one extension portion can comprise threeextension portions, with each of the first, second, and third attachmentpoints being positioned on a respective extension portion.

In exemplary aspects, a disclosed ECM material conduit can comprise anyknown ECM component or material, including, for example and withoutlimitation, mucosal layers and components, submucosal layers andcomponents, muscularis layers and components, and/or basement membranelayers and components. It is contemplated that a disclosed ECM materialconduit can comprise an ECM material obtained from any mammalian tissuesource, including, for example and without limitation, stomach tissue(e.g., stomach submucosa (SS)), small intestinal tissue (e.g., smallintestinal submucosa (SIS)), large intestinal tissue, bladder tissue(e.g., urinary bladder submucosa (UBS)), liver tissue (e.g., liverbasement membrane (LBM)), heart tissue (e.g., pericardium), lung tissue,kidney tissue, pancreatic tissue, prostate tissue, mesothelial tissue,fetal tissue, a placenta, a ureter, veins, arteries, heart valves withor without their attached vessels, tissue surrounding the roots ofdeveloping teeth, and tissue surrounding growing bone. It is furthercontemplated that a disclosed ECM material conduit can comprise an ECMmaterial obtained from ECM components or materials of one or moremammals including, for example and without limitation, humans, cows,pigs, dogs, sheep, cats, horses, rodents, and the like. Thus, it iscontemplated that a disclosed ECM material conduit can comprise ECMcomponents or materials from two or more of the same mammalian species,such as, for example and without limitation, two or more cows, two ormore pigs, two or more dogs, or two or more sheep. It is furthercontemplated that a disclosed ECM material conduit can comprise ECMcomponents or materials from two or more different mammalian species,such as, for example and without limitation, a pig and a cow, a pig anda dog, a pig and a sheep, or a cow and a sheep. It is still furthercontemplated that a disclosed ECM material conduit can comprise ECMcomponents or materials obtained from a first tissue source, such as,for example and without limitation, SIS, from a first mammal, as well asECM components or materials obtained from a second tissue source, suchas, for example and without limitation, SS, from a second mammal.

In various aspects, a disclosed ECM material conduit 10 can be formedfrom a substantially flat sheet of ECM material. In these aspects, theECM material conduit 10 can be formed by securing a first edge of thesheet of ECM material to a second, opposed edge of the sheet of ECMmaterial such that a lumen 12 of the ECM material conduit is defined. Itis contemplated that the first edge of the sheet of ECM material can besecured to the second edge of the sheet of ECM material using anyconventional surgical attachment means, including, for example andwithout limitation, non-absorbable sutures, absorbable sutures, surgicalpastes, surgical glues, staples, and the like. In one exemplary aspect,when non-absorbable sutures are used to secure the first edge of thesheet of ECM material to the second, opposed edge of the sheet of ECMmaterial, it is contemplated that the non-absorbable sutures can bepositioned on an outer surface of the ECM material conduit, therebyreducing the portion of the sutures positioned within the lumen of theECM material conduit. In one optional aspect, it is contemplated thatthe second edge of the sheet of ECM material can be secured inoverlapping relation with the first edge of the sheet of ECM material.In this aspect, it is further contemplated that the portion of the ECMmaterial conduit 10 at which the first and second edges of the sheetoverlap can be everted relative to the lumen of the ECM materialconduit. In another optional aspect, it is contemplated that the secondedge of the sheet of ECM material can be secured in substantialalignment with the first edge of the sheet of ECM material.

In additional aspects, a disclosed ECM material conduit 10 can compriseat least a portion of an intact, lumenal ECM material, such as, forexample and without limitation, a lumenal portion of a native SIS layer.In these aspects, the intact ECM material defines a lumen.

In further aspects, a disclosed ECM material conduit 10 can be formed bygrowing cells, such as, for example and without limitation, fibroblasts,on an outer surface of a cylindrical mandrel using known in vitromethods. In these aspects, it is contemplated that the growth of cellson the outer surface of the mandrel can lead to production of one ormore ECM materials. It is further contemplated that the ECM materialconduit 10 can be decellularized using known methods or as disclosedherein.

In an additional aspect, a disclosed ECM material conduit 10 can belyophilized using known methods. In a further aspect, when a disclosedECM material conduit 10 has been lyophilized, it is contemplated thatthe ECM material conduit can be hydrated using known methods. In thisaspect, it is contemplated that the lyophilized ECM material conduit canbe hydrated in sterile water, saline solution, or a balanced saltsolution for a period ranging from about 5 minutes to about 30 minutes.

Optionally, a disclosed ECM material conduit 10 can be a multi-layerconstruction of two or more layers of ECM material. In one exemplaryaspect, a multi-layer ECM material conduit 10 can be formed from alumenal portion of an intact ECM. As used herein, the term “lumenal”refers to a portion of a material that defines a lumen. In this aspect,the intact lumenal ECM can have a first end and a second end and candefine a lumen. Optionally, the first end of the intact ECM can beinverted into the lumen until it reaches the second end, therebycreating a multi-layer ECM material conduit. Alternatively, the firstend of the intact SIS can be everted over itself until it reaches thesecond end, thereby creating a multi-layer ECM material conduit. In afurther aspect, the multi-layer ECM material conduit can be lyophilizedusing known techniques, thereby creating a multi-laminate ECM materialconduit. In one optional aspect, the multi-layer ECM material conduitcan be positioned over a mandrel during lyophilization. In analternative, optional aspect, during lyophilization of the multi-layerECM material conduit, a cryoballoon can be positioned within the lumenof the multi-layer ECM material conduit and then inflated to presstogether the layers of the multi-layer ECM material conduit. It isfurther contemplated that any conventional lamination method can be usedto laminate the layers of a multi-layer ECM material conduit together,thereby forming a multi-laminate ECM material conduit.

In one aspect, a disclosed ECM material conduit 10 can comprise asterile, acellular ECM composition. In exemplary aspects, such asterile, acellular ECM composition can be formed by contemporaneouslysterilizing and decellularizing an isolated ECM material. Moreparticularly, as disclosed in the following methods, desiredsterilization and decellularization of the isolated ECM material canoccur contemporaneously such that the native properties of the tissuecomposition are maintained and the ECM material is rendered sterile andacellular.

In exemplary aspects, the ECM material conduit 10 can have a multi-layerstructure proximate the inlet and/or outlet portion of the ECM materialconduit. In these aspects, it is contemplated that at least one end ofthe ECM material conduit can be everted or inverted along a portion ofthe length of the ECM material conduit to thereby form a multi-layerstructure proximate the inlet and/or outlet portion of the ECM materialconduit. It is further contemplated that the multi-layer structure caneffectively act as a sewing ring for the ECM material conduit.

Sterilization and Decellularization of ECM Compositions for Use in ECMMaterial Conduits

As described herein, the disclosed methods make use of rapiddepressurization of an isolated ECM material to decellularize the ECMmaterial such that it is acellular. This rapid depressurization of theECM material occurs at depressurization rates that are significantlyhigher than the depressurization rates applied in previously knownmethods. In addition to decellularizing the ECM material as describedherein, the rapid depressurization of the ECM material also can be usedto incorporate desired sterilants and additives into the ECM material.

Optionally, it is contemplated that the ECM material of a disclosed ECMvalve conduit can be sterilized using a known sterilization system, suchas, for example and without limitation, the system described in U.S.Pat. No. 7,108,832, assigned to NovaSterilis, Inc., which patent isexpressly incorporated herein by reference in its entirety. Thus, insome aspects, the system used to perform the disclosed methods cancomprise a standard compressed storage cylinder and a standard aircompressor used in operative association with a booster (e.g., a HaskelBooster AGT 7/30). In other aspects, the air compressor and booster canbe replaced with a single compressor. In exemplary aspects, thecompressed storage cylinder can be configured to receive carbon dioxide,and the booster can be a carbon dioxide booster.

The system can further comprise an inlet port, which allows one or moreadditives contained in a reservoir to be added to a reactor vesselthrough a valve and an additive line. As used herein, the term “reactorvessel” refers to any container having an interior space that isconfigured to receive an ECM material and permit exposure of the ECMmaterial to one or more sterilants and additives, as disclosed herein.In exemplary aspects, the reactor vessel can be, without limitation, abasket, a bucket, a barrel, a box, a pouch, and other known containers.In one aspect, it is contemplated that the reactor vessel can be asyringe that is filled with an ECM material.

It is contemplated that a selected primary sterilant, such as, forexample and without limitation, carbon dioxide, can be introduced to thereactor vessel from a header line via a valve and a supply line. It isfurther contemplated that a filter, such as, for example and withoutlimitation, a 0.5 μm filter, can be provided in the supply line toprevent escape of material from the vessel. In exemplary aspects, apressure gauge can be provided downstream of a shut-off valve in theheader line to allow the pressure to be visually monitored. A checkvalve can be provided in the header line upstream of the valve toprevent reverse fluid flow into the booster. In order to prevent anoverpressure condition existing in the header line, a pressure reliefvalve can optionally be provided.

In one aspect, depressurization of the reactor vessel can beaccomplished using an outlet line and a valve in communication with thereactor vessel. In this aspect, it is contemplated that thedepressurized fluid can exit the vessel via the supply line, be filteredby a filter unit, and then be directed to a separator, where filteredfluid, such as carbon dioxide, can be exhausted via an exhaust line. Itis further contemplated that valves can be incorporated into the variouslines of the apparatus to permit fluid isolation of upstream components.

In one exemplary aspect, the reactor vessel can comprise stainlesssteel, such as, for example and without limitation, 316 gauge stainlesssteel. In another exemplary aspect, the reactor vessel can have a totalinternal volume sufficient to accommodate the materials beingsterilized, either on a laboratory or commercial scale. For example, itis contemplated that the reactor vessel can have a length of about 8inches, an inner diameter of about 2.5 inches, and an internal volume ofabout 600 mL. In additional aspects, the reactor vessel can comprise avibrator, a temperature control unit, and a mechanical stirring systemcomprising an impeller and a magnetic driver. In one optional aspect, itis contemplated that the reactor vessel can contain a basket comprising316 gauge stainless steel. In this aspect, it is contemplated that thebasket can be configured to hold materials to be sterilized while alsoprotecting the impeller and directing the primary sterilant in apredetermined manner.

It is contemplated that the reactor vessel can be operated at a constantpressure or under continual pressurization and depressurization(pressure cycling) conditions without material losses due to splashingor turbulence, and without contamination of pressure lines viaback-diffusion. It is further contemplated that the valves within thesystem can permit easy isolation and removal of the reactor vessel fromthe other components of the system. In one aspect, the top of thereactor vessel can be removed when depressurized to allow access to theinterior space of the reactor vessel.

Optionally, the system can comprise a temperature control unit thatpermits a user to adjustably control the temperature within the reactorvessel.

In use, the disclosed apparatus can be employed in a method of producinga sterilized, acellular ECM composition, such as disclosed herein.However, it is understood that the disclosed apparatus is merelyexemplary, and that any apparatus capable of performing the disclosedmethod steps can be employed to produce the sterilized, acellular ECMcomposition. Thus, the claimed method is in no way limited to aparticular apparatus.

It is contemplated that significant reductions in colony-forming units(CFUs) can be achieved in accordance with the disclosed methods bysubjecting an isolated ECM material to sterilization temperature andpressure conditions using a primary sterilant. Optionally, it iscontemplated that the primary sterilant can be combined with one or moresecondary sterilants to achieve desired sterilization. Optionally, it isfurther contemplated that selected additives can be incorporated into anECM material to impart desired characteristics to the resulting ECMcomposition. It is still further contemplated that the disclosed methodscan be employed to produce sterilized, acellular ECM compositions forimplantation within the body of a subject.

As described herein, the disclosed methods make use of rapiddepressurization of an isolated ECM material to render the ECM materialacellular. This rapid depressurization of the ECM material occurs atdepressurization rates that are significantly higher than thedepressurization rates applied in previously known methods. In additionto rendering acellular the ECM material as described herein, the rapiddepressurization of the ECM material also can be used to enhance theincorporation of desired sterilants and additives into the ECM material.Further, it is contemplated that the rapid depressurization of the ECMmaterial can render the ECM material acellular while also improvingretention of native growth factors, as compared to previously knowndecellularization methods. Still further, it is contemplated that therapid depressurization of the ECM material can be used to improveretention of the tensile strength of the ECM material, as compared topreviously known decellularization methods.

The disclosed methods not only do not significantly weaken themechanical strength and bioptric properties of the ECM compositions, butalso the methods are more effective in decellularizing the ECMcompositions and in enhancing the incorporation of various additivesinto the ECM compositions. Thus, the disclosed sterilization anddecellularization methods provide ECM compositions that are moredecellularized and have a greater capacity to incorporate and thendeliver more additives than ECM compositions known in the art. Moreover,the disclosed sterilization and decellularization methods provide ECMcompositions that have greater amounts and/or concentrations of retainednative growth factors and that have greater tensile strength thansterilized and decellularized ECM compositions known in the art.

In exemplary aspects, the primary sterilant can be carbon dioxide at ornear its supercritical pressure and temperature conditions. However, itis contemplated that any conventional sterilant, including, for example,gas, liquid, or powder sterilants that will not interfere with thenative properties of the ECM material, can be used as the primarysterilant.

In one exemplary aspect, the disclosed sterilization process can bepracticed using carbon dioxide as a primary sterilant at pressuresranging from about 1,000 to about 3,500 psi and at temperatures rangingfrom about 25° C. to about 60° C. More preferably, when supercriticalcarbon dioxide is used, it is contemplated that the sterilizationprocess can use carbon dioxide as a primary sterilant at pressures at orabove 1,071 psi and at temperatures at or above 31.1° C. In this aspect,the ECM material to be sterilized can be subjected to carbon dioxide ator near such pressure and temperature conditions for times ranging fromabout 10 minutes to about 24 hours, more preferably from about 15minutes to about 18 hours, and most preferably, from about 20 minutes toabout 12 hours. Preferably, the carbon dioxide employed in the disclosedsystems and methods can be pure or, alternatively, contain only traceamounts of other gases that do not impair the sterilization propertiesof the carbon dioxide. For ease of further discussion below, the term“supercritical carbon dioxide” will be used, but it will be understoodthat such a term is non-limiting in that carbon dioxide within thepressure and temperature ranges as noted above can be employedsatisfactorily in the practice of the disclosed methods. Within thedisclosed pressure and temperature ranges, it is contemplated that thecarbon dioxide can be presented to the ECM material in a gas, liquid,fluid or plasma form.

The secondary sterilants employed in the disclosed methods can, in someaspects, include chemical sterilants, such as, for example and withoutlimitation, peroxides and/or carboxylic acids. Preferred carboxylicacids include alkanecarboxylic acids and/or alkanepercarboxylic acids,each of which can optionally be substituted at the alpha carbon with oneor more electron-withdrawing substituents, such as halogen, oxygen andnitrogen groups. Exemplary species of chemical sterilants employed inthe practice of the disclosed methods include, for example and withoutlimitation, hydrogen peroxide (H₂O₂), acetic acid (AcA), peracetic acid(PAA), trifluoroacetic acid (TFA), and mixtures thereof. In oneexemplary aspect, the chemical sterilants can include Sporeclenz®sterilant, which is a mixture comprising acetic acid, hydrogen peroxide,and peracetic acid.

It is contemplated that the secondary sterilants can be employed in asterilization-enhancing effective amount of at least about 0.001 vol. %and greater, based on the total volume of the primary sterilant. It isfurther contemplated that the amount of secondary sterilant can bedependent upon the particular secondary sterilant that is employed.Thus, for example, it is contemplated that peracetic acid can be presentin relatively small amounts of about 0.005 vol. % and greater, whileacetic acid can be employed in amounts of about 1.0 vol. % and greater.Thus, it is contemplated that the concentration of the secondarysterilants can range from about 0.001 vol. % to about 2.0 vol. % and cantypically be used as disclosed herein to achieve asterilization-enhancing effect in combination with the disclosed primarysterilants, such as, for example and without limitation, supercriticalcarbon dioxide.

In one aspect, the method of producing a sterilized, acellular ECMcomposition can comprise harvesting a selected tissue from a mammal andrinsing the selected tissue in sterile saline or other biocompatibleliquid, including, for example and without limitation, Ringer's solutionor a balanced biological salt solution. In this aspect, the selectedtissue can be, for example and without limitation, stomach tissue (e.g.,stomach submucosa (SS)), small intestinal tissue (e.g., small intestinalsubmucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinarybladder submucosa (UBS)), liver tissue (e.g., liver basement membrane(LBM)), heart tissue (e.g., pericardium, epicardium, endocardium,myocardium), lung tissue, kidney tissue, pancreatic tissue, prostatetissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins,arteries, heart valves with or without their attached vessels, tissuesurrounding the roots of developing teeth, and tissue surroundinggrowing bone. In another aspect, the method can comprise freezing theselected tissue for a period ranging from about 12 to about 36 hours,more preferably, from about 18 to about 30 hours, and most preferably,from about 22 to about 26 hours. For example, it is contemplated thatthe period during which the selected tissue is frozen can be 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours. 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34hours, 35 hours, 36 hours, and any other period of time falling betweenthe preceding values. In an additional aspect, the method can comprisethawing the selected tissue in cold hypotonic tris buffer. Optionally,in this aspect, the method can comprise thawing the selected tissue incold hypotonic tris buffer on ice with 5 mM ethylenediaminetetraaceticacid (EDTA). In exemplary aspects, it is contemplated that the steps offreezing and thawing the selected tissue can be cyclically repeated upto six times.

In another aspect, the method can comprise isolating an ECM materialfrom the selected tissue. In this aspect, the ECM material can be anymaterial comprising known extracellular matrix components, including,for example and without limitation, stomach tissue (e.g., stomachsubmucosa (SS)), small intestinal tissue (e.g., small intestinalsubmucosa (SIS)), large intestinal tissue, bladder tissue (e.g., urinarybladder submucosa (UBS)), liver tissue (e.g., liver basement membrane(LBM)), heart tissue (e.g., pericardium, epicardium, endocardium,myocardium), lung tissue, kidney tissue, pancreatic tissue, prostatetissue, mesothelial tissue, fetal tissue, a placenta, a ureter, veins,arteries, heart valves with or without their attached vessels, tissuesurrounding the roots of developing teeth, and tissue surroundinggrowing bone, and the like. In one exemplary, non-limiting aspect, thestep of isolating an ECM material can comprise isolating SIS from amammalian tissue source. In this aspect, the method can comprise:incising a wall of a small intestine along a path that is substantiallyparallel to the longitudinal axis of the small intestine; opening thesmall intestine along the path of the incision such that the smallintestine lies flat on a surface; rinsing the small intestine withsterile saline or other biocompatible fluid; mechanically stripping theSIS of the small intestine from the surrounding smooth muscle andserosal layers and from the tunica mucosa, leaving essentially thesubmucosal and basement membrane layers. However, it is contemplatedthat the ECM material can be isolated using any conventional technique,including those described in: U.S. Pat. No. 4,902,508; U.S. Pat. No.5,275,826; U.S. Pat. No. 5,281,422; U.S. Pat. No. 5,554,389; U.S. Pat.No. 6,579,538; U.S. Pat. No. 6,933,326; U.S. Pat. No. 7,033,611;Voytik-Harbin et al., “Identification of Extractable Growth Factors fromSmall Intestinal Submucosa,” J. Cell. Biochem., Vol. 67, pp. 478-491(1997); Hodde et al., “Virus Safety of a Porcine-Derived Medical Device:Evaluation of a Viral Inactivation Method,” Biotech. & Bioeng., Vol. 79,No. 2, pp. 211-216 (2001); Badylak et al., “The Extracellular Matrix asa Scaffold for Tissue Reconstruction,” Cell & Developmental Biology,Vol. 13, pp. 377-383 (2002); Robinson et al., “Extracelular MatrixScaffold for Cardiac Repair,” Circulation, Vol. 112, pp. I-135-I-143(2005); Hodde et al., “Effects of Sterilization on an ExtracellularMatrix Scaffold: Part I. Composition and Matrix Architecture,” J. Mater.Sci.: Mater. Med., Vol. 18, pp. 537-543 (2007); and Hodde et al.,“Effects of Sterilization on an Extracellular Matrix Scaffold: Part II.Bioactivity and Matrix Interaction,” J. Mater. Sci.: Mater. Med., Vol.18, pp. 545-550 (2007), each of which is expressly incorporated hereinby reference in its entirety.

In an additional aspect, the method can comprise incubating the isolatedECM material for 24 to 48 hours in 0.5-1% Triton X-100/0.5-1%Deoxycholic acid with 5 mM EDTA in Dulbecco's Phosphate Buffered Saline(DPBS) (Lonza Walkersville, Inc.). In this aspect, it is contemplatedthat flat or sheet-like ECM materials, such as stomach submucosa (SS),small intestinal submucosa (SIS), and bladder submucosa (UBS), can beincubated in a stretched configuration. It is further contemplated thatECM material conduits or other lumenal ECM materials, such as ureters,arteries, veins, and tubular SIS, can be perfused with the variousdisclosed solutions through soaking and by use of a peristaltic pump.

In a further aspect, after incubation, the method can comprise rinsingthe ECM material with DPBS. In this aspect, it is contemplated that thestep of rinsing the ECM material can comprise rinsing the ECM materialup to six times, including one, two, three, four, five, or six times,with each rinse lasting for about thirty minutes. In an exemplaryaspect, it is contemplated that the step of rinsing the ECM material cancomprise rinsing the ECM material three times, with each rinse lastingfor about thirty minutes.

Optionally, in exemplary aspects, the method can further comprise asecond incubation procedure. In these aspects, the second incubationprocedure can comprise incubating the ECM material in isotonic trisbuffer containing 10-50 μg/mL of RNAase/0.2-0.5 μg/mL DNAase with 5 mMEDTA. It is contemplated that the step of incubating the ECM material inisotonic tris buffer can be performed at a temperature of about 37° C.,substantially corresponding to the temperature of a human body. It isfurther contemplated that the step of incubating the ECM material inisotonic tris buffer can be performed for a period ranging from about 30minutes to about 24 hours, more preferably, from about 1 hour to about18 hours, and most preferably, from about 2 hours to about 12 hours. Inan additional aspect, the second incubation procedure can furthercomprise rinsing the ECM material with DPBS. In this aspect, it iscontemplated that the step of rinsing the ECM material can compriserinsing the ECM material three times, with each rinse lasting for aboutthirty minutes.

In yet another aspect, whether or not the second incubation procedure isperformed, the method can comprise storing the ECM material at atemperature ranging from about 1° C. to about 10° C., more preferably,from about 2° C. to about 6° C., and, most preferably, from about 3° C.to about 5° C. In an exemplary aspect, the ECM material can be stored at4° C.

In an additional aspect, the method can comprise introducing the ECMmaterial into the interior space of the reactor vessel. Optionally, inthis aspect, one or more secondary sterilants from the reservoir can beadded into the interior space of the reactor vessel along with the ECMmaterial. In these aspects, it is contemplated that the one or moresecondary sterilants from the reservoir can be added into the interiorspace of the reactor vessel before, after, or contemporaneously with theECM material. It is further contemplated that the temperature controlunit can be selectively adjusted to produce a desired temperature withinthe interior space of the reactor vessel. In a further aspect, themethod can comprise equilibrating the pressure within the reactor vesseland the pressure within the storage cylinder. For example, in thisaspect, it is contemplated that the pressure within the reactor vesseland the pressure within the storage cylinder can be substantially equalto atmospheric pressure. In yet another aspect, after equilibration ofthe pressures within the apparatus, the method can comprise operatingthe magnetic driver to activate the impeller of the reactor vessel. Instill a further aspect, the method can comprise selectively introducingthe primary sterilant from the storage cylinder into the reactor vesseluntil a desired pressure within the reactor vessel is achieved. In thisaspect, it is contemplated that the step of selectively introducing theprimary sterilant into the reactor vessel can comprise selectivelyactivating the air compressor and the booster to increase flow of theprimary sterilant into the reactor vessel. In exemplary aspects, the aircompressor and booster can be activated to subject the ECM material tosupercritical pressures and temperatures, such as, for example andwithout limitation, the pressures and temperatures necessary to producesupercritical carbon dioxide, for a time period ranging from about 20minutes to about 60 minutes.

In a further aspect, the method can comprise rapidly depressurizing thereactor vessel. In this aspect, a predetermined amount of primarysterilant, such as, for example and without limitation, supercriticalcarbon dioxide, can be released from the reactor vessel through thedepressurization line. It is contemplated that the primary sterilant canbe released from the reactor vessel through opening of the valve coupledto the reactor vessel to thereby rapidly reduce the pressure within thereactor vessel. As used herein, the term “rapid depressurization” refersto depressurization of the reactor vessel at a rate greater than orequal to 400 psi/min. For example, it is contemplated that the reactorvessel can be rapidly depressurized at a depressurization rate rangingfrom about 2.9 MPa/min. to about 18.0 MPa/min. (about 400 psi/min. toabout 2,600 psi/min.), more preferably, from about 5.0 MPa/min. to about10.0 MPa/min. (700 psi/min. to about 1,500 psi/min.), and, mostpreferably, from about 7.0 MPa/min. to about 8.0 MPa/min. (about 1,000psi/min. to about 1,200 psi/min.). Thus, these rapid depressurizationsare significantly greater than the 300 psi/min. depressurization ratedisclosed in U.S. Pat. No. 7,108,832. Without being bound by anyparticular theory, it is believed that the disclosed rapiddepressurization rates increase the level of decellularization achievedin the ECM material. For example, it is contemplated that the rapiddepressurization of a disclosed ECM material can lead to levels ofdecellularization in the ECM material of greater than about 96%,including 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%,97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%,98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%,99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,and 100%.

In exemplary aspects, the method can further comprise the step ofincorporating one or more additives into the ECM material. In theseaspects, it is contemplated that the one or more additives can beprovided in either a powder or a liquid form. In one optional aspect,the step of incorporating the one or more additives can compriseintroducing the one or more additives into the reactor vessel during thestep of rapidly depressurizing the reactor vessel. In this aspect, it iscontemplated that the introduction of the one or more additives can becharacterized as a conventional foaming process. In another optionalaspect, the step of incorporating the one or more additives can compriseintroducing the one or more additives into the reactor vessel after thestep of rapidly depressurizing the reactor vessel. In this aspect, it iscontemplated that the one or more additives can be added to the ECMmaterial after the rapid depressurization of the reactor vessel hascaused the ECM material to swell and/or expand, thereby permittingimproved penetration of the additives into the ECM material. It isfurther contemplated that, in an exemplary aspect, the one or moreadditives can be added to the ECM material within about thirty minutesafter the rapid depressurization of the reactor vessel. In a furtheroptional aspect, the step of incorporating the one or more additives cancomprise introducing the one or more additives into the reactor vesselboth during and after the step of rapidly depressurizing the reactorvessel. In this aspect, it is contemplated that the one or moreadditives can be released into the reactor vessel in both a quick mannerand a slow, extended manner. In still a further optional aspect, thestep of incorporating the one or more additives can comprise introducingthe one or more additives into the reactor vessel before the step ofrapidly depressurizing the reactor vessel.

The disclosed additives can be incorporated into the ECM material toimpart selected properties to the resulting sterilized, acellular ECMcomposition. Thus, it is contemplated that the one or more additives canbe selected to replace or supplement components of the ECM material thatare lost during processing of the ECM material as described herein. Forexample, and as described below, the one or more additives can comprisegrowth factors, cytokines, proteoglycans, glycosaminoglycans (GAGs),proteins, peptides, nucleic acids, small molecules, drugs, or cells. Itis further contemplated that the one or more additives can be selectedto incorporate non-native components into the ECM material. For example,the one or more additives can comprise, for example and withoutlimitation, growth factors for recruiting stem cells, angiogeniccytokines, and anti-inflammatory cytokines. It is still furthercontemplated that the one or more additives can be pharmaceuticalagents, such as statins, corticosteroids, non-steroidalanti-inflammatory drugs, anti-inflammatory compounds, anti-arrhythmicagents, and the like. It is still further contemplated that the one ormore additives can be nanoparticles, such as, for example and withoutlimitation, silver nanoparticles, gold nanoparticles, platinumnanoparticles, iridium nanoparticles, rhodium nanoparticles, palladiumnanoparticles, copper nanoparticles, zinc nanoparticles, and othermetallic nanoparticles. It is still further contemplated that the one ormore additives can be metallic compounds. In one exemplary aspect, theone or more additives can be selected to pharmaceutically suppress theimmune response of a subject following implantation of the resulting ECMcomposition into the body of a subject.

In one aspect, the one or more additives can comprise one or more growthfactors, including, for example and without limitation, transforminggrowth factor-β 1, 2, or 3 (TGF-β 1, 2, or 3), fibroblast growthfactor-2 (FGF-2), also known as basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), placental growth factor(PGF), connective tissue growth factor (CTGF), hepatocyte growth factor(HGF), Insulin-like growth factor (IGF), macrophage colony stimulatingfactor (M-CSF), platelet derived growth factor (PDGF), epidermal growthfactor (EGF), and transforming growth factor-α (TGF-α).

In another aspect, the one or more additives can comprise one or morecytokines, including, for example and without limitation, stem cellfactor (SCF), stromal cell-derived factor-1 (SDF-1), granulocytemacrophage colony-stimulating factor (GM-CSF), interferon gamma(IFN-gamma), Interleukin-3, Interleukin-4, Interleukin-10,Interleukin-13, Leukemia inhibitory factor (LIF), amphiregulin,thrombospondin 1, thrombospondin 2, thrombospondin 3, thrombospondin 4,thrombospondin 5, and angiotensin converting enzyme (ACE).

In an additional aspect, the one or more additives can comprise one ormore proteoglycans, including, for example and without limitation,heparan sulfate proteoglycans, betaglycan, syndecan, decorin, aggrecan,biglycan, fibromodulin, keratocan, lumican, epiphycan, perlecan, agrin,testican, syndecan, glypican, serglycin, selectin, lectican, versican,neurocan, and brevican.

In a further aspect, the one or more additives can comprise one or moreglycosaminoglycans, including, for example and without limitation,heparan sulfate, hyaluronic acid, heparin, chondroitin sulfate B(dermatan sulfate), and chondroitin sulfate A.

In still a further aspect, the one or more additives can comprise one ormore proteins, peptides, or nucleic acids, including, for example andwithout limitation, collagens, elastin, vitronectin, versican, laminin,fibronectin, fibrillin-1, fibrillin-2, plasminogen, small leucine-richproteins, cell-surface associated protein, cell adhesion molecules(CAMs), a matrikine, a matrix metalloproteinase (MMP), a cadherin, animmunoglobin, a multiplexin, cytoplasmic domain-44 (CD-44), amyloidprecursor protein, tenascin, nidogen/entactin, fibulin I, fibulin II,integrins, transmembrane molecules, and osteopontin.

In yet another aspect, the one or more additives can comprise one ormore pharmaceutical agents, including, for example and withoutlimitation, statin drugs, for example, cerevastatin, atorvastatin,fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin, corticosteroids, non-steroidalanti-inflammatory drugs, anti-inflammatory compounds, anti-arrhythmicagents, antimicrobials, antibiotics, and the like.

In exemplary aspects, the steps of introducing the one or more additivesinto the reactor vessel can comprise opening the valve to allow the oneor more additives to flow from the reservoir into the inlet port. Priorto pressurization, it is contemplated that the one or more additives canbe introduced directly into the reactor vessel prior to sealing and/orvia the inlet port.

It is contemplated that the disclosed rapid depressurization andrepressurization of the reactor vessel, with or without the addition ofthe one or more additives, can be repeated for any desired number ofcycles. It is further contemplated that the cycles of depressurizationand repressurization, as well as the introduction of the primarysterilants and/or secondary sterilants and/or additives, can beautomatically controlled via a controller that is configured toselectively open and/or close the various valves of the system toachieve desired pressure conditions and cycles.

In some aspects, the disclosed methods can further comprise the step ofagitating the contents of the reactor vessel. In these aspects, it iscontemplated that the step of agitating the contents of the reactorvessel can comprise periodically agitating the contents of the reactorvessel using a vibrator. It is further contemplated that the agitationof the reactor vessel can be intermittent, continual, or continuous. Inexemplary aspects, the step of agitating the contents of the reactorvessel can occur during the step of introducing the primary sterilantinto the reactor vessel. It is contemplated that the agitation of thecontents of the reactor vessel can enhance the mass transfer of thesterilants and/or additives by eliminating voids in the fluids withinthe reactor vessel to provide for more complete contact between the ECMmaterial and the sterilants and/or additives. It is further contemplatedthat the step of agitating the contents of the reactor vessel cancomprise selectively adjusting the intensity and duration of agitationso as to optimize sterilization times, temperatures, andpressurization/depressurization cycles.

In a further aspect, after the sterilization and decellularization ofthe ECM material is complete, the method can further comprisedepressurizing the reactor vessel and deactivating the magnetic drive soas to cease movement of the stirring impeller. Finally, the method cancomprise the step of removing the resulting sterilized, acellular ECMcomposition through the top of the reactor vessel.

Methods of Regenerating Heart Valves Using the ECM Material Conduits

Also disclosed herein are methods of regenerating heart valves. In anexemplary aspect, a method of regenerating an atrioventricular (AV)valve to replace a defective AV valve within a heart of a subject isdisclosed. In this aspect, and with reference to FIG. 1, it iscontemplated that the defective AV valve is attached at an annulusbetween an atrium and a ventricle within the heart of the subject and isfunctionally coupled to a plurality of papillary muscles 32 within theventricle of the heart of the subject. As used herein, the term“atrioventricular (AV) valve” can refer to either a mitral (bi-cuspid)valve or a tri-cuspid valve within the heart of the subject. It iscontemplated that, if the defective AV valve is a mitral valve, then thedefective AV valve is attached at an annulus between the left atrium andthe left ventricle of the heart of the subject. It is furthercontemplated that, if the defective AV valve is a tri-cuspid valve, thenthe defective AV valve is attached at an annulus between the rightatrium and the right ventricle of the heart of the subject.

In one aspect, and with reference to FIG. 2, a disclosed method ofregenerating an AV valve can comprise removing the defective AV valvefrom the heart of the subject, thereby exposing an annular region 30 ofthe heart of the subject. As used herein, the term “annular region”refers to the portion of the heart of a subject that is proximate to thenative position of an annulus between an atrium and a ventricle withinthe heart of the subject. When an annulus is positioned within the heartof the subject, the annular region 30 includes the annulus as well asthe heart muscle proximate the annulus. When the annulus has beenremoved from the heart of the subject, the annular region 30 includesthe heart muscle proximate the former position of the annulus within theheart of the subject.

In one optional aspect, it is contemplated that the annulus of theannular region 30 can be removed from the heart of the subject alongwith the defective AV valve. In another optional aspect, it iscontemplated that the chordae tendineae that are coupled to thedefective AV valve can be removed from the heart of the subject alongwith the defective AV valve. It is contemplated that the step ofremoving the defective AV valve can further comprise placing the subjecton cardiopulmonary bypass. It is further contemplated that the step ofremoving the defective AV valve can further comprise arresting and/orfibrillating the heart of the subject and exposing the defective valvethrough an incision in an atrium of the heart of the subject.

In an additional aspect, a disclosed method of regenerating an AV valvecan further comprise implanting an ECM material conduit 10, such asthose disclosed herein. In this aspect, and as further disclosed herein,the ECM material conduit 10 can define a lumen 12 and have an inletportion 14 and an outlet portion 18, with each of the inlet portion andthe outlet portion having an outer circumference. It is contemplatedthat the lumen 12 defined by the ECM material conduit 10 can have acenter point 13 proximate the outlet portion 18 of the ECM materialconduit. In a further aspect, the step of implanting an ECM materialconduit 10 can comprise securing the inlet portion 14 of the ECMmaterial conduit to the annular region 30 and securing the outletportion of the ECM material conduit to at least two of the plurality ofpapillary muscles 32. In this aspect, it is contemplated that, when theannulus is not removed from the heart of the subject, the inlet portion14 of the ECM material conduit 10 can be secured to the annulus. It isfurther contemplated that, when the annulus is removed from the heart ofthe subject, the inlet portion 14 of the ECM material conduit 10 can besecured to heart muscle proximate the native location of the excisedannulus. It is still further contemplated that the ECM material conduit10 can be secured to the annular region 30 and/or the papillary muscles32 using any conventional surgical attachment means, including, forexample and without limitation, non-absorbable sutures, absorbablesutures, surgical pastes, surgical glues, staples, and the like.Optionally, in one aspect, the ECM material conduit 10 can be secured tothe papillary muscles 32 before it is secured to the annular region. Inthis aspect, it is contemplated that, after the ECM material conduit 10has been properly secured to the papillary muscles 32, the length of theECM material conduit 10 along the longitudinal axis 24 can be trimmed asnecessary to eliminate any excess length while retaining adequate tissuefor proper attachment of the ECM material conduit to the annular region30. Alternatively, in another aspect, the ECM material conduit 10 can besecured to the annular region 30 before it is secured to the papillarymuscles 32. In this aspect, it is contemplated that, after the ECMmaterial conduit 10 has been properly secured to the annular region 30,the length of the ECM material conduit along the longitudinal axis 24can be trimmed as necessary to eliminate any excess length whileretaining adequate tissue for proper attachment of the ECM materialconduit to the papillary muscles 32.

Optionally, in one aspect, and with reference to FIGS. 2-3, thedisclosed method can be used to regenerate a bi-cuspid AV valve withinthe heart of the subject. In this aspect, the step of implanting an ECMmaterial conduit 10 can comprise securing the outlet portion 18 of theECM material conduit to only two papillary muscles (i.e., only a firstpapillary muscle 32 a and a second papillary muscle 32 b) of theplurality of papillary muscles. As one will appreciate, the leftventricle only has two papillary muscles, whereas the right ventriclehas three papillary muscles. Nonetheless, it is contemplated that,regardless of whether the defective AV valve is a mitral valve or atri-cuspid valve, the replacement AV valve regenerated by the ECMmaterial conduit will be a bi-cuspid valve. When the defective AV valveis a tri-cuspid valve, it is contemplated that the first papillarymuscle 32 a can be the anterior papillary muscle of the right ventricleand that the second papillary muscle 32 b can be the posterior papillarymuscle of the right ventricle. It is further contemplated that the firstpapillary muscle 32 a can be the anterior papillary muscle of the rightventricle and that the second papillary muscle 32 b can be the septalpapillary muscle of the right ventricle. It is still furthercontemplated that the first papillary muscle 32 a can be the posteriorpapillary muscle of the right ventricle and that the second papillarymuscle 32 b can be the septal papillary muscle of the right ventricle.

In a further aspect, the step of implanting an ECM material conduit cancomprise securing the outlet portion of the ECM material conduit 10 tothe first papillary muscle 32 a at a first attachment point 34 a and toa second papillary muscle 32 b at a second attachment point 34 b. Inthis aspect, it is contemplated that the second attachment point 34 bcan be spaced from the first attachment point 34 a along the outercircumference of the outlet portion 18 of the ECM material conduit 10.In yet another aspect, and with reference to FIG. 3, it is contemplatedthat the center point 13 of the lumen 12 of the ECM material conduit 10can correspond to a vertex of an angle 40 formed between the firstattachment point 34 a and the second attachment point 34 b. In thisaspect, it is further contemplated that the angle 40 formed between thefirst attachment point and the second attachment point can have adesired magnitude. In an exemplary aspect, the desired magnitude of theangle 40 formed between the first attachment point 34 a and the secondattachment point 34 b can range from about 120° to about 150°. In thisaspect, and as shown in FIG. 3, it is contemplated that the angle 40formed between the first attachment point 34 a and the second attachmentpoint 34 b can have a complementary angle 42 within the lumen 12. It isfurther contemplated that the complementary angle 42 within the lumen 12can have a magnitude ranging from about 210° to about 240°.

In exemplary aspects, when the outlet portion 18 of the ECM materialconduit 10 comprises at least one extension portion as described herein,it is contemplated that one or more of the first and second attachmentpoints 34 a, 34 b can be positioned on a corresponding extension portionof the at least one extension portion. In these aspects, it iscontemplated that the at least one extension portion can comprise twoextension portions, with each of the first and second attachment points34 a, 34 b being positioned on a respective extension portion.

Optionally, in another aspect, the disclosed method can be used toregenerate a tri-cuspid AV valve within the heart of the subject. Inthis aspect, when the defective valve is the tri-cuspid valve, the stepof implanting an ECM material conduit 10 can comprise securing theoutlet portion 18 of the ECM material conduit to each of the threepapillary muscles within the right ventricle. In a further aspect, thestep of implanting an ECM material conduit 10 can comprise securing theoutlet portion 18 of the ECM material conduit to a first papillarymuscle at a first attachment point, to a second papillary muscle at asecond attachment point, and to a third papillary muscle at a thirdattachment point. In this aspect, it is contemplated that the first,second, and third attachment points can be spaced from one another alongthe outer circumference of the outlet portion of the ECM materialconduit. In an exemplary aspect, it is contemplated that the first,second, and third attachment points can optionally be substantiallyequally spaced along the outer circumference of the outlet portion ofthe ECM material conduit. However, it is contemplated that the spacingof the first, second, and third attachment points can vary dependingupon the native anatomy and positioning of the papillary muscles. It iscontemplated that, in exemplary aspects, where the first, second, andthird attachment points can be spaced such that 40% of the operativecircumference of the ECM material conduit is between the septal andanterior papillary muscles, 30% of the circumference of the ECM materialconduit is between the anterior and posterior papillary muscles, and 30%of the circumference of the ECM material conduit is between theposterior and septal papillary muscles. In exemplary aspects, when theoutlet portion 18 of the ECM material conduit 10 comprises at least oneextension portion as described herein, it is contemplated that one ormore of the first, second, and third attachment points can be positionedon a corresponding extension portion of the at least one extensionportion. In these aspects, it is contemplated that the at least oneextension portion can comprise three extension portions, with each ofthe first, second, and third attachment points being positioned on arespective extension portion.

After the ECM material conduit 10 is properly secured to the annularregion and to the papillary muscles, and after any necessary trimming orsculpting of the ECM material conduit has been completed, the atrium ofthe heart of the subject can be closed and the heart of the subject canbe restarted.

It is contemplated that, following implantation of the ECM materialconduit as disclosed herein, the ECM material conduit can becomepopulated with cells from the subject that will gradually remodel theECM material of the ECM material conduit into heart valve tissue that isidentical or substantially identical to properly functioning nativeheart valve tissue. It is further contemplated that stem cells canmigrate to the ECM material conduit from the points at which the ECMmaterial conduit is attached to the papillary muscles and the annularregion within the heart of the subject. It is still further contemplatedthat, during circulation of epithelial and endothelial progenitor cells,the surfaces of the ECM material conduit can rapidly become lined orcovered with epithelial and/or endothelial progenitor cells. It is stillfurther contemplated that the points at which the ECM material conduitis attached to the papillary muscles and the annular region can serve aspoints of constraint that direct the remodeling of the ECM material intoleaflet tissue or chordae tendineae that are identical or substantiallyidentical to properly functioning native leaflet tissue and properlyfunctioning native chordae tendineae. It is still further contemplatedthat, where the annulus is removed from the annular region prior toattachment of the ECM material conduit, the inlet portion of the ECMmaterial conduit can direct the remodeling of an annulus that isidentical or substantially identical to a properly functioning nativeannulus.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example One

Exemplary valve dimensions were measured for both porcine and humanvalves as shown in FIG. 4 and Table 1. These measurements supported theuse of ECM material conduits having a 30 mm annular diameter and alength ranging from about 32-35 mm. These design criteria also suggestedthat the distal valve should be implanted so as to achieve 40% of thecircumference between the septal and anterior papillary muscles, 30%between the anterior and posterior papillary muscles, and 30% betweenthe posterior and septal papillary muscles.

TABLE 1 Leaflet Porcine Human Weight (g) 376 ± 188 380 ± 180 Annularlength (mm) Anterior 46.57 ± 8.53  40.00 ± 6.71  Septal 39.15 ± 9.00 32.04 ± 5.71  Posterior 34.21 ± 7.55  28.94 ± 5.40  Thickness (mm)Anterior 0.390 ± 0.102 0.396 ± 0.101 Septal 0.378 ± 0.106 0.380 ± 0.086Posterior 0.379 ± 0.105 0.413 ± 0.079 Commissure height [mm] Anterior7.80 ± 2.02 11.16 ± 1.49* Septal 6.73 ± 1.58 13.36 ± 2.16* Posterior6.69 ± 1.36 13.25 ± 2.22* Leaflet maximum height Anterior 19.93 ± 3.53 24.96 ± 3.12* (mm) Septal 18.82 ± 3.22  21.54 ± 5.31  Posterior 18.62 ±4.08  21.40 ± 2.69 

Example Two

In vitro hemodynamic evaluation of an exemplary ECM material conduit wasachieved in a physiologic right heart simulator. These studiesdemonstrated that when a valve construct 30 mm diameter and 35 mm inlength was implanted so that the papillary muscle attachment wasperformed so that 40% of the circumference was between the septal andanterior papillary muscles, 30% was between the anterior and posterior,and 30% was between the posterior and septal, the valve opened andclosed with a low transvalvular pressure gradient and closing volume andno regurgitation. An image of the closed valve in the in vitro flow loopis shown in FIG. 5. These studies demonstrated that the valve wasfunctional under physiologic conditions.

In vitro mechanical evaluation was also performed on the tricuspid valveseam (n=8) and the maximum tensile break force for the sewn seam wasfound to be 52.1±14.1 N (11.7±3.16 lb_(f)) with a minimum and maximum of34.8 N (7.82 lb_(f)), and 72.3 N (16.25 lb_(f)), respectively. Thetensile force, ball burst, and suture pull-out forces for the 4-ply ECMconduit was determined to be 19.35±5.51 N (4.35±1.24 lb_(f)), 126.6±30.2N (6699±1598 mmHg), and 11.12±2.08 N (2.50±0.47 lb_(f)), respectively.These values are more than adequate to meet the force requirements forthis valve in the low-pressure environment of the right heart.

Example Three

In a non-GLP study using a sheep model, the tricuspid valve in foursheep was replaced by a 2-ply ECM valve conduit as described herein. Theprocedure was successfully completed on all four animals. All of theanimals were euthanized on schedule at 3, 5, 8, and 12 monthspost-implant. Echocardiographic results (Table 2) for all four animalsshowed that the mobility and function of the leaflets appear normal,with only a mild level of backflow through the valve after closure. Thevalves were seen to experience normal forward flow with mildregurgitation. Necropsy results showed that the replacement valvesappeared to be grossly within normal limits and at 12 months theleaflets remodeled and appear similar to native valve tissue.

TABLE 2 Animal #/ Assessment Implant Time Point Regur- Date (weeks)gitation Comments 2948 0 Mild Mobility and function of the leafletsappear (Jan. 13, 2011) 1 Mild normal. Leaflet coaptation lookssufficient and 4 Mild annulus appears morphologically normal. 12 MildNormal forward flow with mild to trivial regurgitation 2981 0 MildMobility and function of the leaflets appears (Feb. 14, 2011) 1 Mildnormal. Leaflet coaptation looks sufficient and 4 Mild annulus appearsmorphologically normal. 20 Mild to Normal forward flow with a mild levelof Moderate regurgitation. 2969 0 Mild Mobility and function of theleaflets appears (Mar. 10, 2011) 20 None normal. Leaflet coaptationlooks sufficient and 24 Mild annulus appears morphologically normal. 32Mild Normal forward flow with a mild to no regurgitation. 2966 0 NoneMobility and function of the leaflets appears (Feb. 10, 2011) 1 Mildnormal. Leaflet coaptation looks sufficient and 4 None annulus appearsmorphologically normal. 24 None Normal forward flow with noregurgitation 32 None noted except at the one-week time point. 40 None48 None

The replacement valves appeared grossly similar to the native valve thatwas replaced. FIG. 7 shows 3, 5, 8, and 12-month explants from sheepimplanted with the ECM Material Conduit.

Echocardiography showed good hemodynamics for the valves out to 12months. Some of these animals exhibited a mild level of backflow throughthe valve after closure, which was recorded by the echocardiographytechnician as “mild” or “mild to moderate”. Echocardiography showedcomplete coaptation of the leaflets with no leaflet prolapse (FIG. 6).The degree of apparent valvular insufficiency in these tubularprosthetic valves is exaggerated on echocardiography because of theresidual fluid that is trapped within the cylinder when the valves areopen. This “closing volume” fluid may be ejected retrograde upon valveclosure, which on ECHO would appear to be regurgitant volume when thereis no actual regurgitation present.

FIG. 7 shows images of the valve as seen from the right atrium in theclosed position demonstrate the progressive remodeling that is occurringover time in the sheep. At the 3-month time point (FIG. 7( a)),remodeling has already occurred at the valve annulus and is extending tothe leaflets to regenerate apparently normal valve tissue. At 12 months(FIG. 7( d)), the leaflets have remodeled and appear similar to nativevalve tissue. Similarly, as shown in FIG. 8, the papillary muscleattachment points are remodeled almost completely at 3 months.

Histology from the sheep at 3, 5, 8, and 12 months followingimplantation of the CorMatrix ECM Tricuspid Valve stained with a MovatPentachrome stain was performed. At 3 months, elastin formation wasalready evident at the valve annulus, and at 5 months elastin was alsobeing generated at the papillary muscle attachment region. By 8 months,the majority of the ECM had been resorbed and remodeled into hosttissue. Cells were distributed throughout the valve and the remodeledtissue had formed a three-layer structure similar to the native valvetissue with elastin in the outer layers and GAGs in the middle.

Example Four

In exemplary applications of the disclosed sterilization anddecellularization methods, selected tissues were harvested and rinsed insterile saline. The selected tissues were then frozen for 24 hours. Thefrozen tissues were thawed in cold hypotonic tris buffer on ice with 5mM ethylenediaminetetraacetic acid (EDTA). An extracellular matrixmaterial was then isolated from each selected tissue, as describedherein.

The isolated extracellular matrix materials were incubated for 24 to 48hours in 0.5-1% Triton X-100/0.5-1% Deoxycholic acid with 5 mM EDTA inDulbecco's Phosphate Buffered Saline (DPBS) (Lonza Walkersville, Inc.).Flat extracellular matrix materials, such as stomach submucosa (SS),small intestinal submucosa (SIS), and bladder submucosa (UBS), wereincubated in a stretched configuration. Tubular extracellular matrixmaterials, such as ureters, arteries, veins, and tubular SIS, wereperfused with the solutions through soaking and by use of a peristalticpump.

After incubation, each extracellular matrix material was rinsed threetimes with DPBS. Each rinsing with DPBS lasted 30 minutes. Someextracellular matrix materials were then incubated for 2 to 12 hours at37° C. in isotonic tris buffer containing 10-50 μg/mL of RNAse/0.2-0.5μg/mL DNAse with 5 mM EDTA. Following this incubation step, theextracellular matrix materials were again rinsed three times with DPBS.Each rinsing with DPBS lasted 30 minutes. The extracellular matrixmaterials were stored at 4° C.

Within 48 hours of storage, the extracellular matrix materials wereprocessed in supercritical carbon dioxide as disclosed herein for 20-60minutes at temperatures at or greater than 31.1° C. and pressures at orgreater than 1,071 psi. After this sterilization step, the extracellularmatrix materials were rapidly depressurized at a rate of 2.7 MPa/10 sec.(391.6 psi/10 sec.) for a minute and 19 seconds. During this time, thepressure applied to the extracellular matrix materials rapidly decreasedfrom 9.9 MPa to 0.69 MPa.

The extracellular matrix materials were then processed in supercriticalcarbon dioxide and peracetic acid (PAA) as disclosed herein for 30minutes to 6 hours to achieve terminal sterilization. In this processingstep, the pressure applied to the extracellular matrix materials wasincreased to 9.9 MPa. The resulting sterilized, acellular extracellularmatrix materials were then packaged in Tyvek® (E.I. du Pont de Nemours &Company) pouches that were sealed within plastic pouches to preventfluid leakage.

Table 3 summarizes the sterilization and decellularization of porcineureter, bovine pericardium, and porcine mesothelium.

TABLE 3 Deoxy- Triton cholic TX- RNAse/ Supercritical X-100 Acid100/Deoxy DNAse CO₂/PAA Material Conc. Conc. incubation incubation timePorcine 0.5% 0.5% 24 hours 2 hours 120 minutes ureters Bovine 0.5% 0.5%24 hours 2 hours 180 minutes pericardium Porcine 0.5% 0.5% 24 hours 2hours 120 minutes mesothelium

Example Five

The DNA content of ECM material samples was measured as an indicator ofdecellularization of the respective ECM material samples using varioussterilization and decellularization techniques. The measured DNA contentwas evaluated with a pico green assay in which DNA was labeled with afluorescent label that was detected with a spectrophotometer. Themeasured DNA content was normalized by the dry weight of the samples.DNA content was measured and evaluated for the following treatmentgroups: (1) Lyophilized, non-sterile SIS; (2) Ethylene Oxide(EtO)-sterilized SIS; (3) Lyophilized, non-sterile SIS that wassterilized through a 60 minute treatment with PAA and supercritical CO₂,as disclosed herein; (4) Lyophilized, non-sterile SIS that wassterilized through a 20 minute treatment with PAA and supercritical CO₂,as disclosed herein; and (5) Raw, unprocessed SIS.

FIG. 9 shows the total DNA content for the respective samples, asnormalized by dry weight. FIG. 10 shows the percent of DNA that wasremoved from each respective sample, as compared to raw, unprocessedSIS. These results indicated that by sterilizing the non-sterile SISusing a 60 minute treatment with PAA and supercritical CO₂, as disclosedherein, over 96% of the DNA found in raw SIS was removed, as compared toonly 94% when the SIS was sterilized by EtO and only 93% when the SISwas not sterilized by any method.

Example Six

Ureters were processed with a gentle detergent (0.5% Triton X-100/0.5%Sodium Deoxycholate in 5 mM EDTA in DPBS) for 24 hours and then rinsedthree times in DPBS as disclosed herein. After this pretreatment, theureters were decellularized and sterilized using rapid depressurizationand treatment with PAA and supercritical CO₂, as disclosed herein.Hematoxylin and Eosin (H&E) Stains were prepared for one sample ureterat the following stages of treatment: (A) native ureter; (B) pretreatedureter; and (C) pretreated ureter with rapid depressurization andtreatment with PAA and supercritical CO₂, as disclosed herein. Thesestains indicated that DNA content was significantly reduced with rapiddepressurization.

Example Seven

The growth factor content of ECM material samples was measured.Enzyme-linked immunosorbent (ELISA) assays were performed on the ECMmaterial samples to quantify the content of bFGF and the active form ofTGF-β in each respective sample. The following treatment groups wereevaluated: (1) Lyophilized, non-sterile SIS; (2) Ethylene Oxide(EtO)-sterilized SIS; (3) Lyophilized, non-sterile SIS that wassterilized through a 60 minute treatment with PAA and supercritical CO₂,as disclosed herein; (4) Lyophilized, non-sterile SIS that wassterilized through a 20 minute treatment with PAA and supercritical CO₂,as disclosed herein; and (5) Raw, unprocessed SIS. The bFGF content andTGF-β content measurements were normalized by dry weight of eachrespective sample. These results are shown in FIGS. 11 and 12. Theseresults indicated that the concentration of both growth factors wasreduced by exposure to EtO. However, the concentration of the growthfactors was not affected by sterilization with PAA and supercriticalCO₂.

Example Eight

Using the methods disclosed herein, supercritical CO₂ was used as aprimary sterilant and as a carrier for adding bFGF into SIS sheets.First, the respective SIS sheets were placed into Tyvek® pouches alongwith varying amounts of bFGF. The pouches were exposed to supercriticalCO₂ for 60 minutes at 9.6 MPa. The pouches were rapidly depressurized ata rate of 7.20 MPa/min. Samples were directly processed in 16 mL PAA insupercritical CO₂ for 20 minutes. The following treatment groups wereevaluated: (1) No bFGF added; (2) 5 μL bFGF added; and (3) 15 μL bFGFadded. Each μL of bFGF contained 0.1 μg of bFGF. Thus, since each SISsheet weighed approximately 0.5 g, the maximum concentrations of bFGFfor the 5 μL and 15 μL groups were about 4170 pg/mg dry weight and about12,500 pg/mg dry weight, respectively. The bFGF content for these groupsis shown in FIG. 13, as measured with respect to the dry weight of therespective samples. These results indicated that the measuredconcentrations of bFGF did not reach the maximum concentrations and thatthe sample to which 15 μL of bFGF was added did not have a measuredconcentration of bFGF that was three times greater than the measuredconcentration of bFGF in the sample to which 5 μL of bFGF was added.

Example Nine

The tensile strengths of two-ply SIS samples were measured. Thefollowing treatment groups were evaluated: (1) EtO Treatment; (2)PAA/supercritical CO₂ treatment for 20 minutes; (3) PAA/supercriticalCO₂ treatment for 60 minutes; and (4) PAA/supercritical CO₂ treatmentfor 120 minutes. The tensile strength test results are shown in FIG. 14.These results indicated that the SIS samples that were processed withPAA/supercritical CO₂ for 20 or 120 minutes, as disclosed herein, weresignificantly stronger than the SIS samples that were processed withEtO.

Example Ten

Rapid depressurization was used following gentle detergent soaks orperfusion of the ECM materials listed in Table 4 (below) at the notedconcentrations and for the noted time periods. Tissues were harvestedand rinsed in saline. The tissues were frozen for at least 24 hours. Thetissues were thawed in cold hypotonic tris buffer on ice with 5 mM EDTA.The ECM of interest was isolated. For flat tissues (e.g., stomachsubmucosa, small intestine submucosa, and bladder submucosa), the tissuewas stretched on a tissue stretching device and incubated in solutionsin a stretched configuration. For tubular tissues (e.g., ureters,arteries, veins, and tubular SIS), the tissue was perfused withsolutions using a peristaltic pump and were soaked during incubation.The tissues were incubated for 2 to 24 hours in 0.5% Triton X-100/0.5%Deoxycholic acid with 5 mM EDTA in DPBS. The tissues were rinsed 3 timesfor 15-30 minutes each time in DPBS. The tissues were stored at 4° C.Within 48 hours of tissue storage, the tissues were processed insupercritical CO₂ for 20-120 minutes followed by rapid depressurization(RDP)(decrease in pressure from 9.9 MPa to 0.69 MPa in 1 min 19 sec,corresponding to a depressurization of 2.7 MPa/10 sec).

TABLE 4 Triton X-100 Deoxycholic TX-100/Deoxy Supercritical CO₂ MaterialConc. Acid Conc. incubation time Porcine 0.5% 0.5% 24 hours 60 minutesureters Bovine 0.5% 0.5% 24 hours 60 minutes pericardium Porcine 0.5%0.5%  2 hours 60 minutes mesothelium SIS 0.5% 0.5%  2 hours 60 minutes

The results showed that supercritical CO₂ exposure followed by rapiddepressurization (SCCO₂+RDP) did aid in the removal of cell remnants andDNA while preserving growth factors in the ECMs.

Example Eleven

The growth factor content of various ECM compositions was analyzed usingbasic fibroblast growth factor (bFGF) as a representative growth factor.bFGF was selected because it is a prevalent growth factor in native ECMtissues. An enzyme-linked immunosorbent assay (ELISA, R&D Systems,Minneapolis, Minn.) was used to measure the bFGF content in thefollowing samples: (1) Unprocessed (Raw) SIS; (2) SIS after detergentsoak (TX-deoxy) only; (3) SIS after TX-deoxy and RDP (includes SCCO₂);(4) SIS after TX-deoxy, RDP, and PAA (SCCO₂ with PAA for sterilization);(5) SIS after TX-deoxy, and PAA; (6) SIS sterilized by EtO (supplied byCook Biotech, Inc.); and (7) non-sterile SIS (supplied by Cook Biotech,Inc.).

In these studies, SIS was used to compare an ECM composition processedwith and without RDP to SIS provided by Cook Biotech, Inc. Some of theprocessed SIS was also sterilized using the described SCCO₂+PAA methodafter decellularization. The measured growth factor content of therespective ECM compositions is shown in FIG. 15.

These results indicate that the rapid depressurization process was moreeffective than other decellularization processes at preserving the bFGFcontent and that the additional RDP processing to remove residual DNAand cell fragments results in only a small loss of bFGF. By comparison,the PAA sterilization process appeared to remove almost all of theremaining bFGF, even in the absence of RDP. Additionally, the rapiddepressurization process preserved more of the bFGF content in thenative SIS than the Cook decellularization methods. For purposes ofthese results, when the bFGF content was reduced, it is assumed that allother growth factor content was similarly reduced since the growthfactors are all bound to the ECM compositions in a similar manner.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. An ECM valve conduit for regenerating anatrioventricular (AV) valve to replace a defective AV valve within aheart of a subject, the AV valve being attached to an annular region ofthe heart. the defective AV valve and annular region of the heartcomprising first cardiovascular tissue, the defective AV valve furthercomprising a first plurality of valve leaflets, comprising: abioabsorbable tubular conduit member comprising sterile, acellular smallintestine submucosa (SIS), said SIS comprising less than 4% DNA contentand a dry weight bFGF content of at least 140 pg/mg of said SIS, saidSIS further exhibiting at least 96% decellurization and a tensilestrength of at least 9 N, said ECM conduit member defining a lumen andhaving an inlet portion and an outlet portion, said inlet portion ofsaid ECM conduit member being configured to attach to an said annularregion of said heart of said subject, said outlet portion of said ECMconduit member being configured to attach to papillary muscles withinsaid subject's heart, said ECM conduit member being configured to inducegeneration of a plurality of regenerated valve leaflets in said ECMconduit member lumen when said ECM conduit member is coupled to saidfirst cardiovascular tissue of said annular region, said plurality ofregenerated valve leaflets comprising second cardiovascular tissue, saidsecond cardiovascular tissue and said first cardiovascular tissue beingsimilar, said plurality of regenerated valve leaflets being configuredto function in a manner similar to said first valve leaflets of saiddefective AV valve.
 2. The ECM valve conduit of claim 1, wherein saidinlet portion and said outlet portion of said ECM conduit haverespective outer circumferences, and wherein said outer circumference ofsaid outlet portion is equal to said outer circumference of said inletportion.
 3. The ECM valve conduit of claim 1, wherein said outletportion of said ECM conduit member includes a first extension portionand a second extension portion.
 4. The ECM valve conduit of claim 3,wherein said first and second extension portions are spaced at 0° and120°, respectively, on said ECM conduit member outlet portion.
 5. TheECM valve conduit of claim 3, wherein said first and second extensionportions are spaced at 0° and 150°, respectively, on said ECM conduitmember outer portion.
 6. The ECM valve conduit of claim 5, wherein atleast a portion of said ECM conduit comprises a multi-laminatestructure.